CA1227131A - Diagnostic test for streptococcus a - Google Patents
Diagnostic test for streptococcus aInfo
- Publication number
- CA1227131A CA1227131A CA000466006A CA466006A CA1227131A CA 1227131 A CA1227131 A CA 1227131A CA 000466006 A CA000466006 A CA 000466006A CA 466006 A CA466006 A CA 466006A CA 1227131 A CA1227131 A CA 1227131A
- Authority
- CA
- Canada
- Prior art keywords
- antigen
- swab
- streptococcus
- antibody
- occurrence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 241000194017 Streptococcus Species 0.000 title claims abstract description 57
- 238000002405 diagnostic procedure Methods 0.000 title abstract description 4
- 239000000427 antigen Substances 0.000 claims abstract description 58
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 50
- 102000036639 antigens Human genes 0.000 claims abstract description 48
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- 238000000605 extraction Methods 0.000 claims abstract description 31
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- 239000004033 plastic Substances 0.000 claims abstract description 7
- 229920003023 plastic Polymers 0.000 claims abstract description 7
- 241000187759 Streptomyces albus Species 0.000 claims abstract 2
- 238000012360 testing method Methods 0.000 claims description 36
- 239000004816 latex Substances 0.000 claims description 21
- 229920000126 latex Polymers 0.000 claims description 21
- 239000002245 particle Substances 0.000 claims description 16
- 239000012472 biological sample Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 230000004520 agglutination Effects 0.000 claims description 11
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- 229920003043 Cellulose fiber Polymers 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 241000187747 Streptomyces Species 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- 229920002994 synthetic fiber Polymers 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 2
- 241001505901 Streptococcus sp. 'group A' Species 0.000 abstract description 8
- 229920000297 Rayon Polymers 0.000 abstract description 6
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- 208000015181 infectious disease Diseases 0.000 description 3
- -1 polypep-tone Substances 0.000 description 3
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- 201000007100 Pharyngitis Diseases 0.000 description 2
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- 206010039587 Scarlet Fever Diseases 0.000 description 1
- 102100036788 Tubulin beta-4A chain Human genes 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
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- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Air Bags (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
DIAGNOSTIC TEST FOR STREPTOCOCCUS A
ABSTRACT OF THE DISCLOSURE
The presence of Group A Streptococcus in a biological specimen is determined from the presence of Streptococcus A antigen. A biological specimen is collected with an applicator having a plastic stick with a rayon swab. The swab is placed in an extraction reagent containing enzymes produced by the bacterium Streptomyces albus, wherein the enzymes release the antigen from the fiber. An aliquot of the extraction medium is mixed with an indicator reagent containing an antibody reactive with the antigen. The occurrence or non-occurrence of an antibody-antigen reaction is noted which indicates the presence or absence of Group A Streptococcus in the biological specimen.
ABSTRACT OF THE DISCLOSURE
The presence of Group A Streptococcus in a biological specimen is determined from the presence of Streptococcus A antigen. A biological specimen is collected with an applicator having a plastic stick with a rayon swab. The swab is placed in an extraction reagent containing enzymes produced by the bacterium Streptomyces albus, wherein the enzymes release the antigen from the fiber. An aliquot of the extraction medium is mixed with an indicator reagent containing an antibody reactive with the antigen. The occurrence or non-occurrence of an antibody-antigen reaction is noted which indicates the presence or absence of Group A Streptococcus in the biological specimen.
Description
lZ~7~31 DIAGNOSTIC TEST FOR STREPTOCOCCUS A
The present invention relates to the detection of infectious agents through an antibody-antigen reaction and more particularly to the detection of Streptococcus A through the presence in a biological sample of Streptococcus A antigen.
BACKGROUND OF THE INVENTION
Of the several groups of Streptococci, group A
streptococcus (S. pudginess) is primarily responsible for causing pathological conditions in humans, such as B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac suckle, glomerulonephritis, septic sore throat, and puerperal sepsis. Other groups of Streptococci are wholly innocuous and normally exist, for example, in the throat. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence in an early stage of infection so that an appropriate course of treatment may be selected.
Streptococci may be cultured conventionally in suitable media; however, identifying Streptococci by type is not a simple task. Streptococci A-selective culture media are less than perfect in that they are not fully selective, i.e., they do not eliminate all other types of Streptococci while allowing Stewart-coccus A to grow. Importantly, culturing techniques for identifying Streptococci A generally require an incubation time of 18 hours, and frequently as long as 48 hours, to ascertain the presence of Stewart-coccus A. Such lengthy tests delay a fully informed 1, 12Z7~
The present invention relates to the detection of infectious agents through an antibody-antigen reaction and more particularly to the detection of Streptococcus A through the presence in a biological sample of Streptococcus A antigen.
BACKGROUND OF THE INVENTION
Of the several groups of Streptococci, group A
streptococcus (S. pudginess) is primarily responsible for causing pathological conditions in humans, such as B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac suckle, glomerulonephritis, septic sore throat, and puerperal sepsis. Other groups of Streptococci are wholly innocuous and normally exist, for example, in the throat. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence in an early stage of infection so that an appropriate course of treatment may be selected.
Streptococci may be cultured conventionally in suitable media; however, identifying Streptococci by type is not a simple task. Streptococci A-selective culture media are less than perfect in that they are not fully selective, i.e., they do not eliminate all other types of Streptococci while allowing Stewart-coccus A to grow. Importantly, culturing techniques for identifying Streptococci A generally require an incubation time of 18 hours, and frequently as long as 48 hours, to ascertain the presence of Stewart-coccus A. Such lengthy tests delay a fully informed 1, 12Z7~
-2-judgment as to the best course of disease treatment.
The desirability of a reliable, simple and quick test for Streptococcus A is clearly indicated.
SUMMARY OF THE INVENTION
Testing for the presence of Streptococcus A in a biological sample, such as a saliva sample from the throat, is quick and reliable using a test kit provided by the present invention. An applicator, including a stick and a Streptococcus A antigen collecting-fiber swab at one end, is used to swab an infected area. After a sample is taken with the swab, the swab is dipped in an extraction reagent that releases the antigen from the swab fibers into the reagent. An Alcott of the extraction reagent is introduced into an indicator solution that con-twins an antibody reactive to the antigen. Occur-fence or non-occurrence of an antibody-antigen reaction is indicative of the presence or absence of Streptococcus A in the biological sample.
Thus in one embodiment the present invention provides a test kit for the detection of Streptococcus A comprising applicator means for collecting a biological sample potentially containing Streptococcus A, said applicator means including an applicator stick and a swab at one end formed of a fiber that collects Streptococcus A
antigen, an extraction reagent containing enzymes for releasing Streptococcus A antigen from said swab, and indicator reagent containing antibody reactive with Streptococcus A antigen, whereby when a biological sample is collected with said fiber swab of said applicator means, said swab is placed in said extraction reagent for a time sufficient to release Streptococcus A antigen into said extraction no--pa- ~2Z7~31 agent, and an Alcott of said extraction reagent is mixed with said indicator reagent, the presence of Streptococcus A in said biological sample is indicated by the occurrence or non-occurrence of an antibody-antigen reaction.
In another embodiment the invention provides a method for detecting Streptococcus A comprising providing an applicator including an applicator stick and a fibrous swab and swabbing a biological sample 10 with said swab, providing an extraction reagent containing enzymes for effecting release of Streptococcus A antigen from said swab, dipping said swab in said extraction reagent and incubating said swab within said extraction reagent for a period of time sufficient to release antigen from said fibrous swab, providing indicator reagent containing antibody reactive with said antigen and adding an Alcott of said extraction reagent thereto, and noting the occurrence or non-occurrence of an antibody-antigen reaction, indicating the presence or absence of Streptococcus A in said biological sample.
Several features of the invention have been found to promote reliable results. Regenerated cellulose fiber, commonly known as rayon, is most effective for collecting and releasing (in the presence of enzymes) Streptococcus A antigen, whereas natural cellulosic fibers, e.g. cotton, do not work well. It is preferable that the stick be formed of a non-porous material, most preferably a synthetic, non-porous material such as a non-porous plastic rather than a natural material, such as wood. The ~227~3~
preferred extraction reagent contains an enzyme mixture produced by the bacterium Streptomyces album. The preferred indicator reagent is an aglow-Tunisian reagent in which antibody that is reactive with the Streptococcus A antigen is bound to latex particles so that the particles agglutinate as a result of an antigen-antibody reaction.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Provided herein is a diagnostic test for group A Streptococcus which can be performed in a very short time, i.e., less than about 70 minutes, and without the use of complicated equipment. This permits the test to be performed in a doctor's office and enables the doctor to determine a course of treatment based upon the results of the test the same day. The test detects the presence of Stewart-coccus A antigen in a biological sample, such as a swab specimen from the throat, rather than growth of the Streptococcus A organism itself, as is done in culture tests. Hence, the extended incubation period required for selective culturing tests is substantially eliminated. The advantages of the invention; however, are not obtained at the expense of either sensitivity or accuracy as studies have shown that both sensitivity and accuracy of the assay of the invention test approach 100%.
In accordance with the invention, a test kit provides the materials and reagents by which the presence of Streptococcus A antigen in a biological ~227~3~
specimen is accurately detected. A specimen is collected by means of an applicator that includes an applicator stick and a fiber swab at one end of the stick. The area of infection is swabbed with the fiber swab, whereby Streptococcus A antigen is collected by the fibers. Subsequently, the swab is dipped in an aqueous extraction reagent containing a mixture of enzymes produced by the bacterium Stewart-maces album and which effect release of Streptococcus A antigen from the swab. An indicator reagent is provided that contains antibody that is specifically reactive with the Streptococcus A antigen. When the extraction reagent containing Streptococcus A antigen (if Streptococcus A antigen is present in the boo-logical sample) is added to the indicator reagent, detectable antibody-antigen reaction occurs.
An important feature of the invention is the use of the extraction reagent containing enzymes that effect release of Streptococcus A antigen from a swab. It has been found that merely swabbing an infected area with a swab and placing the swab in a non-enzyme containing solution does not provide reliable results. It is believed that the Stewart-coccus A antigen tends to cling to the fibers of the swab rather than dissolving freely into an aqueous medium which does not contain enzymes. It is found, however, that in the presence of an enzyme mixture obtained from Streptomyces album, Streptococcus A
antigen is released into an aqueous medium. The mechanism of antigen release is unknown; however, it is believed that the enzymes release that portion ~227~31 of the Streptococcus A antigen molecule containing the antibody-reactive determinant(s) from that portion of the molecule that tends to cling to the swab fibers.
extraction reagent containing Streptomyces album enzymes is prepared as follows:
Streptomyces album (NCTC #7807) is grown at 25C
for 5 days in Mycophil (Trademark) ajar obtained from the BLUE Microbiology Division of Beckon, Dickinson loan Company, Cockeysville, Maryland. The organisms are then transferred into 40 ml of liquid medium and allowed to grow for 5 days at 25C, with shaking.
The liquid medium consists of Yeast Extract, polypep-tone, dextrose, polysorbate 80 (Tweet), and potassium 15monobasic phosphate.
After 5 days, the 40 ml of liquid medium is transferred to 400 ml of the same medium and incus bated at 25C with shaking. Between 5 and 14 days allocates of the culture broth are taken and evaluated pharaoh their ability to lyre Streptococcus pudginess (ATTICS #8135) cells.
The culture fluid is harvested by centrifugation at 10,000-13,000 x g for 30 minutes. The superannuate is then filtered through cheesecloth and dialyzed against 0.05 M Trip pi 8Ø Allocates are then lyophilized and kept at 4C.
Furthermore, it is found that the correct choice of an apply icator is an important factor affecting test reliability. Many common types off applicators used for gathering specimens do not work well in the test of the present invention. In ~7:~31 particular, swabs formed of natural cellulosic material are found to work very poorly. In accord dance with an important aspect of the invention, it is highly preferable that the culture swab be formed of regenerated cellulose rayon) fibers. Rayon is preferred not only over cotton but over other Cynthia-tic fibers which are otherwise useful for forming culture swabs.
Another important discovery with respect to the present invention is that applicator sticks made of natural material, such as wood, are not suitable for obtaining good results. In accordance with another important aspect of the invention, it is preferred that the applicator sticks be formed of synthetic material, especially material that is non-porous, including non-porous plastics such as polystyrene, polycarbonate, polypropylene, polyethylene, polyp tetrafluoroethelene, polyamides and polyacrylics.
It is surprising that regenerated cellulose fiber is most effective for collecting Streptococcus A antigen whereas natural cellulosic materials, such as cotton and wood, are unsuitable. This finding indicates that the system is very sensitive to the presence of substances which are present along with cellulose in natural materials. The mechanism or mechanisms which cause this sensitivity have not been determined; however, several factors may be involved. It may be that natural cellulosic ma-trials contain substances which inhibit the enzymes that are used to release the Streptococcus A antigen from the fibers. Another possibility is that natural cellulosic fibers contain substances which react with 12~7~3~ P- 744 the antigenically active sites of the Streptococcus A antigen, rendering the antigen unrecognizable by the antibody. Porous material, such as wood, may further interfere with clear results by absorbing the antigen, enzymes or other reagents. The possible mechanisms by which natural cellulosic materials interfere with detection of Streptococcus A antigen are set forth as possible explanations for the surprisingly superior results achieved using Cynthia-lo tic materials; however, because the possible explant-lions have not been closely examined, applicants are not bound by any such possible explanations for the results.
In accordance with a preferred embodiment of the invention, presence of Streptococcus A antigen in the extraction reagent is detected by an agglutina-lion indicator reagent that contains suspended latex particles to which the antigen-reactive antibody is bound. Binding of antigen molecules by antibodies bound to different latex particles results in cross-linking of the latex particles, causing them to agglutinate and precipitate out of suspension.
Agglutination can be detected with the naked eye, but preferably is noted by examination with a microscope.
Suitable latex particles may be purchased from Polysciences, Inc., Barrington, Pennsylvania in carboxylated form. The particles range in size from about .2 to about 1.0 microns and typically have between about 1 x 101 and about 1 x 1012 anti-body molecules per cm2 of particle surface area.
12~:7~31 The indicator reagent has from between about 1 x 10 3 and about 10 x 10 3 gym of antibody-bound latex particles per ml. The antibody for preparing the particles are preferably obtained by immunizing rabbits with Strain T-23 Group A beta-hemolytic streptococci. Active antibody is recovered from the serum of these rabbits by a combination of precipita-lion in the presence of 50% saturated (NH4)2SO4 and ion exchange chromatography using DEAE-cellulose DOW, Whitman) equilibrated with Trip buffer, 0.01M
pi 8Ø The active antibody fraction is eluded from the DEAR cellulose column by the use of a Nail gradient. The active antibody fractions elude from the column at between 0.04 and 0.09 M Nail.
15Latex-antibody reagent is prepared by coupling rabbit anti-Group A Streptococcus antibody to car-boxylated latex using 1-ethyl-3-(3-dimethyl amino-propel) carbodiimide (ED) as the condensing agent.
It should be understood that the invention is not limited to detection of Streptococcus A through agglutination of antibody-bound latex particles, and numerous other suitable methods of detecting anti-body-antigen reactions could be substituted, in-eluding but not limited to radio immunoassay and immunofluorescent techniques. An advantage of the immunoagglutination technique is that it is simple to perform, gives results within minutes and is clearly readable.
A test kit according to the invention provides suitable applicators with plastic sticks and rayon swabs. Enzyme-containing extraction reagent is lZ~7~L31 typically provided in lyophilized form for on-site reconstitution. After reconstitution, the extraction reagent may be stored refrigerated (about 2C to about 8C) for about 10 days. The indicator reagent is provided as a stable suspension and can be stored refrigerated for about 12 months. The suspension is shaken prior to use. A test kit also preferably provides additional supplies for performing the test to insure that the most suitable supplies are used lo and to prevent the use of materials which might interfere with the test. Such supplies can include test tubes of appropriate size in which antigen is extracted from the swabs, multi-well test plates for performance of the agglutination reaction, hand stirring sticks, transfer pipette tips, etc. A test kit should also provide positive (Streptococcus A
antigen-containing) and negative control samples. It is also preferred that as a further control, the test kit includes a latex suspension that contains no antibodies for direct comparison (bearing in mind that even a non-agglutinated suspension appears somewhat cloudy).
Reconstituted extraction reagent is pipette into clean test tubes (typically about 0.5 ml per tube). The applicators are used to swab infected areas, e.g., throats, and then the swab end of each stick is placed in one of the tubes. The tubes are incubated for at least 30 minutes and preferably an hour at about body temperature (37 C) to allow the enzyme to release the Streptococcus A from the fiber.
During incubation, it is preferred that the top of ~.~27~3~
--1 o--the test tube be covered by any suitable means to prevent evaporation. After the incubation period, the applicator swab is pressed against the sides of the tube to release its liquid as the applicator is withdrawn. The applicator is then discarded. An Alcott, e.g., 50 us, of each sample is placed in each of two wells of a sample plate, as are allocates of positive and negative controls to provide three sets of paired wells. To one of the paired wells is added antibody-containing latex suspension and to the other of the two wells is added antibody-free latex suspension. Using individual plastic stirring sticks, the reagents in each well are mixed. The sample plate is covered and placed on a mechanical rotator for about 4 minutes. The results are immediately readable. Although a strong agglutina-lion reaction is visible to the naked eye, it is preferred that the mixtures in the wells be observed under a microscope. Any difference observed in the antibody-containing latex suspension well from the antibody-free latex suspension well indicates the presence of Streptococcus A in the swabbed area. If there is no difference in the two wells, it can be concluded that Streptococcus A is not present.
4.0 ml of carboxylated latex having 2.5 weight percent solids, obtained from Polysciences, Inc., Barrington, PA, is washed three times with distilled water. After the final wash the particles are resuspended in 4.0 ml of distilled water. One ml of 12Z~13~
0.05 M KH2PO4, pi 4.5 is added. The suspension of latex is placed in a magnetic stirrer and main-twined at 22C. 5.0 ml of a solution of 2 weight percent ED (obtained from Sigma Chemical Company, St. Louis, MO) is added and allowed to react for 3.5 his. The carbodiimide latex is washed once in saline (0.9% Nail) and resuspended in 5.0 ml of saline (0.9%
Nail).
1.2 my of rabbit antibody is dissolved in 5.0 ml of 0.2M borate, pi 8.5, and the 5.0 ml of anti-voted latex suspension in saline is added. The latex and rabbit antibody are allowed to react for 20 his.
at 22C. To neutralize surface carboxyl groups not bound to the antibody, a solution of 5 my ethanol famine is added, followed by a solution of bovine serum albumin at a concentration of 2 weight percent.
The antibody-latex is washed and taken up in 0.1 M
Gleason pi 8.2 containing 0.9% Nail, 0.2% Nan, 0.2% BRA, and 0.05% Tony, and is stored at 4C.
The performance of the Group A Streptococcus latex agglutination test described above was deter-mined in a multi-center clinical evaluation. The latex agglutination test results were compared to culture results.
Pharyngeal swabs were collected from 1440 adults and children who exhibited the symptoms of pharyngitis. Specimens were collected on a rayon swab and transported to the laboratory in Modified ~27~3~ P- 744 Stuart's Medium (Marion Culturette). Prior to the performance of the assay of the invention, each swab was used to inoculate a sheep blood ajar plate for culture. After 18-24 hours of incubation, beta-5 humility to colonies were grouped by the capillaryprecipitin test. The latex agglutination test method of the invention agreed with the culture results in 95% (1366/1440) of the swab specimens.
The results are summarized in Table 1.
lo AGREEMENT BETWEEN THE
AGGLUTINATION TEST AND CULTURE RESULTS
Agglutination Agglutination Culture Results Positive Negative Positive for Group - 309 281 28 lo A Streptococcus Negative for Group - 1131 46 1085 A Streptococcus The sensitivity of the Group A Streptococcus latex agglutination test was determined from 309 20 pharyngeal swab specimens which were determined to be positive by culture for beta-hemolytic streptococci and confirmed as Group A by the capillary precipitin :1227~L31.
test. Two hundred eighty-one (281) of these specie miens (91%) were positive by the latex agglutination test of the invention. The sensitivity of the test was 95% if those swabs which grew out less than 10 colonies of Group A Streptococcus on culture were ignored.
The specificity of the Group A Streptococcus latex agglutination test was determined from 1067 pharyngeal swabs yielding cultures negative for beta-hemolytic streptococci and from 64 pharyngeal swabs yielding cultures which grew a beta-hemolytic streptococcus confirmed by the capillary precipitin test as being other than Group A. Of these, 1085 samples were agglutination negative, yielding a specificity of 96%.
Although the invention has been described in terms of certain preferred embodiments, modifications obvious to one with ordinary skill in the art may be made without departing from the scope of the invention.
Various features are set forth in the following claims.
The desirability of a reliable, simple and quick test for Streptococcus A is clearly indicated.
SUMMARY OF THE INVENTION
Testing for the presence of Streptococcus A in a biological sample, such as a saliva sample from the throat, is quick and reliable using a test kit provided by the present invention. An applicator, including a stick and a Streptococcus A antigen collecting-fiber swab at one end, is used to swab an infected area. After a sample is taken with the swab, the swab is dipped in an extraction reagent that releases the antigen from the swab fibers into the reagent. An Alcott of the extraction reagent is introduced into an indicator solution that con-twins an antibody reactive to the antigen. Occur-fence or non-occurrence of an antibody-antigen reaction is indicative of the presence or absence of Streptococcus A in the biological sample.
Thus in one embodiment the present invention provides a test kit for the detection of Streptococcus A comprising applicator means for collecting a biological sample potentially containing Streptococcus A, said applicator means including an applicator stick and a swab at one end formed of a fiber that collects Streptococcus A
antigen, an extraction reagent containing enzymes for releasing Streptococcus A antigen from said swab, and indicator reagent containing antibody reactive with Streptococcus A antigen, whereby when a biological sample is collected with said fiber swab of said applicator means, said swab is placed in said extraction reagent for a time sufficient to release Streptococcus A antigen into said extraction no--pa- ~2Z7~31 agent, and an Alcott of said extraction reagent is mixed with said indicator reagent, the presence of Streptococcus A in said biological sample is indicated by the occurrence or non-occurrence of an antibody-antigen reaction.
In another embodiment the invention provides a method for detecting Streptococcus A comprising providing an applicator including an applicator stick and a fibrous swab and swabbing a biological sample 10 with said swab, providing an extraction reagent containing enzymes for effecting release of Streptococcus A antigen from said swab, dipping said swab in said extraction reagent and incubating said swab within said extraction reagent for a period of time sufficient to release antigen from said fibrous swab, providing indicator reagent containing antibody reactive with said antigen and adding an Alcott of said extraction reagent thereto, and noting the occurrence or non-occurrence of an antibody-antigen reaction, indicating the presence or absence of Streptococcus A in said biological sample.
Several features of the invention have been found to promote reliable results. Regenerated cellulose fiber, commonly known as rayon, is most effective for collecting and releasing (in the presence of enzymes) Streptococcus A antigen, whereas natural cellulosic fibers, e.g. cotton, do not work well. It is preferable that the stick be formed of a non-porous material, most preferably a synthetic, non-porous material such as a non-porous plastic rather than a natural material, such as wood. The ~227~3~
preferred extraction reagent contains an enzyme mixture produced by the bacterium Streptomyces album. The preferred indicator reagent is an aglow-Tunisian reagent in which antibody that is reactive with the Streptococcus A antigen is bound to latex particles so that the particles agglutinate as a result of an antigen-antibody reaction.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Provided herein is a diagnostic test for group A Streptococcus which can be performed in a very short time, i.e., less than about 70 minutes, and without the use of complicated equipment. This permits the test to be performed in a doctor's office and enables the doctor to determine a course of treatment based upon the results of the test the same day. The test detects the presence of Stewart-coccus A antigen in a biological sample, such as a swab specimen from the throat, rather than growth of the Streptococcus A organism itself, as is done in culture tests. Hence, the extended incubation period required for selective culturing tests is substantially eliminated. The advantages of the invention; however, are not obtained at the expense of either sensitivity or accuracy as studies have shown that both sensitivity and accuracy of the assay of the invention test approach 100%.
In accordance with the invention, a test kit provides the materials and reagents by which the presence of Streptococcus A antigen in a biological ~227~3~
specimen is accurately detected. A specimen is collected by means of an applicator that includes an applicator stick and a fiber swab at one end of the stick. The area of infection is swabbed with the fiber swab, whereby Streptococcus A antigen is collected by the fibers. Subsequently, the swab is dipped in an aqueous extraction reagent containing a mixture of enzymes produced by the bacterium Stewart-maces album and which effect release of Streptococcus A antigen from the swab. An indicator reagent is provided that contains antibody that is specifically reactive with the Streptococcus A antigen. When the extraction reagent containing Streptococcus A antigen (if Streptococcus A antigen is present in the boo-logical sample) is added to the indicator reagent, detectable antibody-antigen reaction occurs.
An important feature of the invention is the use of the extraction reagent containing enzymes that effect release of Streptococcus A antigen from a swab. It has been found that merely swabbing an infected area with a swab and placing the swab in a non-enzyme containing solution does not provide reliable results. It is believed that the Stewart-coccus A antigen tends to cling to the fibers of the swab rather than dissolving freely into an aqueous medium which does not contain enzymes. It is found, however, that in the presence of an enzyme mixture obtained from Streptomyces album, Streptococcus A
antigen is released into an aqueous medium. The mechanism of antigen release is unknown; however, it is believed that the enzymes release that portion ~227~31 of the Streptococcus A antigen molecule containing the antibody-reactive determinant(s) from that portion of the molecule that tends to cling to the swab fibers.
extraction reagent containing Streptomyces album enzymes is prepared as follows:
Streptomyces album (NCTC #7807) is grown at 25C
for 5 days in Mycophil (Trademark) ajar obtained from the BLUE Microbiology Division of Beckon, Dickinson loan Company, Cockeysville, Maryland. The organisms are then transferred into 40 ml of liquid medium and allowed to grow for 5 days at 25C, with shaking.
The liquid medium consists of Yeast Extract, polypep-tone, dextrose, polysorbate 80 (Tweet), and potassium 15monobasic phosphate.
After 5 days, the 40 ml of liquid medium is transferred to 400 ml of the same medium and incus bated at 25C with shaking. Between 5 and 14 days allocates of the culture broth are taken and evaluated pharaoh their ability to lyre Streptococcus pudginess (ATTICS #8135) cells.
The culture fluid is harvested by centrifugation at 10,000-13,000 x g for 30 minutes. The superannuate is then filtered through cheesecloth and dialyzed against 0.05 M Trip pi 8Ø Allocates are then lyophilized and kept at 4C.
Furthermore, it is found that the correct choice of an apply icator is an important factor affecting test reliability. Many common types off applicators used for gathering specimens do not work well in the test of the present invention. In ~7:~31 particular, swabs formed of natural cellulosic material are found to work very poorly. In accord dance with an important aspect of the invention, it is highly preferable that the culture swab be formed of regenerated cellulose rayon) fibers. Rayon is preferred not only over cotton but over other Cynthia-tic fibers which are otherwise useful for forming culture swabs.
Another important discovery with respect to the present invention is that applicator sticks made of natural material, such as wood, are not suitable for obtaining good results. In accordance with another important aspect of the invention, it is preferred that the applicator sticks be formed of synthetic material, especially material that is non-porous, including non-porous plastics such as polystyrene, polycarbonate, polypropylene, polyethylene, polyp tetrafluoroethelene, polyamides and polyacrylics.
It is surprising that regenerated cellulose fiber is most effective for collecting Streptococcus A antigen whereas natural cellulosic materials, such as cotton and wood, are unsuitable. This finding indicates that the system is very sensitive to the presence of substances which are present along with cellulose in natural materials. The mechanism or mechanisms which cause this sensitivity have not been determined; however, several factors may be involved. It may be that natural cellulosic ma-trials contain substances which inhibit the enzymes that are used to release the Streptococcus A antigen from the fibers. Another possibility is that natural cellulosic fibers contain substances which react with 12~7~3~ P- 744 the antigenically active sites of the Streptococcus A antigen, rendering the antigen unrecognizable by the antibody. Porous material, such as wood, may further interfere with clear results by absorbing the antigen, enzymes or other reagents. The possible mechanisms by which natural cellulosic materials interfere with detection of Streptococcus A antigen are set forth as possible explanations for the surprisingly superior results achieved using Cynthia-lo tic materials; however, because the possible explant-lions have not been closely examined, applicants are not bound by any such possible explanations for the results.
In accordance with a preferred embodiment of the invention, presence of Streptococcus A antigen in the extraction reagent is detected by an agglutina-lion indicator reagent that contains suspended latex particles to which the antigen-reactive antibody is bound. Binding of antigen molecules by antibodies bound to different latex particles results in cross-linking of the latex particles, causing them to agglutinate and precipitate out of suspension.
Agglutination can be detected with the naked eye, but preferably is noted by examination with a microscope.
Suitable latex particles may be purchased from Polysciences, Inc., Barrington, Pennsylvania in carboxylated form. The particles range in size from about .2 to about 1.0 microns and typically have between about 1 x 101 and about 1 x 1012 anti-body molecules per cm2 of particle surface area.
12~:7~31 The indicator reagent has from between about 1 x 10 3 and about 10 x 10 3 gym of antibody-bound latex particles per ml. The antibody for preparing the particles are preferably obtained by immunizing rabbits with Strain T-23 Group A beta-hemolytic streptococci. Active antibody is recovered from the serum of these rabbits by a combination of precipita-lion in the presence of 50% saturated (NH4)2SO4 and ion exchange chromatography using DEAE-cellulose DOW, Whitman) equilibrated with Trip buffer, 0.01M
pi 8Ø The active antibody fraction is eluded from the DEAR cellulose column by the use of a Nail gradient. The active antibody fractions elude from the column at between 0.04 and 0.09 M Nail.
15Latex-antibody reagent is prepared by coupling rabbit anti-Group A Streptococcus antibody to car-boxylated latex using 1-ethyl-3-(3-dimethyl amino-propel) carbodiimide (ED) as the condensing agent.
It should be understood that the invention is not limited to detection of Streptococcus A through agglutination of antibody-bound latex particles, and numerous other suitable methods of detecting anti-body-antigen reactions could be substituted, in-eluding but not limited to radio immunoassay and immunofluorescent techniques. An advantage of the immunoagglutination technique is that it is simple to perform, gives results within minutes and is clearly readable.
A test kit according to the invention provides suitable applicators with plastic sticks and rayon swabs. Enzyme-containing extraction reagent is lZ~7~L31 typically provided in lyophilized form for on-site reconstitution. After reconstitution, the extraction reagent may be stored refrigerated (about 2C to about 8C) for about 10 days. The indicator reagent is provided as a stable suspension and can be stored refrigerated for about 12 months. The suspension is shaken prior to use. A test kit also preferably provides additional supplies for performing the test to insure that the most suitable supplies are used lo and to prevent the use of materials which might interfere with the test. Such supplies can include test tubes of appropriate size in which antigen is extracted from the swabs, multi-well test plates for performance of the agglutination reaction, hand stirring sticks, transfer pipette tips, etc. A test kit should also provide positive (Streptococcus A
antigen-containing) and negative control samples. It is also preferred that as a further control, the test kit includes a latex suspension that contains no antibodies for direct comparison (bearing in mind that even a non-agglutinated suspension appears somewhat cloudy).
Reconstituted extraction reagent is pipette into clean test tubes (typically about 0.5 ml per tube). The applicators are used to swab infected areas, e.g., throats, and then the swab end of each stick is placed in one of the tubes. The tubes are incubated for at least 30 minutes and preferably an hour at about body temperature (37 C) to allow the enzyme to release the Streptococcus A from the fiber.
During incubation, it is preferred that the top of ~.~27~3~
--1 o--the test tube be covered by any suitable means to prevent evaporation. After the incubation period, the applicator swab is pressed against the sides of the tube to release its liquid as the applicator is withdrawn. The applicator is then discarded. An Alcott, e.g., 50 us, of each sample is placed in each of two wells of a sample plate, as are allocates of positive and negative controls to provide three sets of paired wells. To one of the paired wells is added antibody-containing latex suspension and to the other of the two wells is added antibody-free latex suspension. Using individual plastic stirring sticks, the reagents in each well are mixed. The sample plate is covered and placed on a mechanical rotator for about 4 minutes. The results are immediately readable. Although a strong agglutina-lion reaction is visible to the naked eye, it is preferred that the mixtures in the wells be observed under a microscope. Any difference observed in the antibody-containing latex suspension well from the antibody-free latex suspension well indicates the presence of Streptococcus A in the swabbed area. If there is no difference in the two wells, it can be concluded that Streptococcus A is not present.
4.0 ml of carboxylated latex having 2.5 weight percent solids, obtained from Polysciences, Inc., Barrington, PA, is washed three times with distilled water. After the final wash the particles are resuspended in 4.0 ml of distilled water. One ml of 12Z~13~
0.05 M KH2PO4, pi 4.5 is added. The suspension of latex is placed in a magnetic stirrer and main-twined at 22C. 5.0 ml of a solution of 2 weight percent ED (obtained from Sigma Chemical Company, St. Louis, MO) is added and allowed to react for 3.5 his. The carbodiimide latex is washed once in saline (0.9% Nail) and resuspended in 5.0 ml of saline (0.9%
Nail).
1.2 my of rabbit antibody is dissolved in 5.0 ml of 0.2M borate, pi 8.5, and the 5.0 ml of anti-voted latex suspension in saline is added. The latex and rabbit antibody are allowed to react for 20 his.
at 22C. To neutralize surface carboxyl groups not bound to the antibody, a solution of 5 my ethanol famine is added, followed by a solution of bovine serum albumin at a concentration of 2 weight percent.
The antibody-latex is washed and taken up in 0.1 M
Gleason pi 8.2 containing 0.9% Nail, 0.2% Nan, 0.2% BRA, and 0.05% Tony, and is stored at 4C.
The performance of the Group A Streptococcus latex agglutination test described above was deter-mined in a multi-center clinical evaluation. The latex agglutination test results were compared to culture results.
Pharyngeal swabs were collected from 1440 adults and children who exhibited the symptoms of pharyngitis. Specimens were collected on a rayon swab and transported to the laboratory in Modified ~27~3~ P- 744 Stuart's Medium (Marion Culturette). Prior to the performance of the assay of the invention, each swab was used to inoculate a sheep blood ajar plate for culture. After 18-24 hours of incubation, beta-5 humility to colonies were grouped by the capillaryprecipitin test. The latex agglutination test method of the invention agreed with the culture results in 95% (1366/1440) of the swab specimens.
The results are summarized in Table 1.
lo AGREEMENT BETWEEN THE
AGGLUTINATION TEST AND CULTURE RESULTS
Agglutination Agglutination Culture Results Positive Negative Positive for Group - 309 281 28 lo A Streptococcus Negative for Group - 1131 46 1085 A Streptococcus The sensitivity of the Group A Streptococcus latex agglutination test was determined from 309 20 pharyngeal swab specimens which were determined to be positive by culture for beta-hemolytic streptococci and confirmed as Group A by the capillary precipitin :1227~L31.
test. Two hundred eighty-one (281) of these specie miens (91%) were positive by the latex agglutination test of the invention. The sensitivity of the test was 95% if those swabs which grew out less than 10 colonies of Group A Streptococcus on culture were ignored.
The specificity of the Group A Streptococcus latex agglutination test was determined from 1067 pharyngeal swabs yielding cultures negative for beta-hemolytic streptococci and from 64 pharyngeal swabs yielding cultures which grew a beta-hemolytic streptococcus confirmed by the capillary precipitin test as being other than Group A. Of these, 1085 samples were agglutination negative, yielding a specificity of 96%.
Although the invention has been described in terms of certain preferred embodiments, modifications obvious to one with ordinary skill in the art may be made without departing from the scope of the invention.
Various features are set forth in the following claims.
Claims (14)
1. A test kit for the detection of Strepto-coccus A comprising applicator means for collecting a biological sample potentially containing Streptococcus A, said applicator means including an applicator stick and a swab at one end formed of a fiber that collects Streptococcus A antigen, an extraction reagent containing enzymes for releasing Streptococcus A antigen from said swab, and indicator reagent containing antibody reactive with Streptococcus A antigen, whereby when a biological sample is collected with said fiber swab of said applicator means, said swab is placed in said extraction reagent for a time sufficient to release Streptoeoecus A antigen into said extraction reagent, and an aliquot of said extraction reagent is mixed with said indicator reagent, the presence of Streptococcus A in said bio-logical sample is indicated by the occurrence or non-occurrence of an antibody-antigen reaction.
2. A test kit according to Claim 1 wherein said swab is formed of regenerated cellulose fiber.
3. A test kit according to Claim 1 wherein said stick is formed of synthetic material.
4. A test kit according to Claim 1 wherein said stick is formed of non-porous material.
5. A test kit according to Claim 1 wherein said swab is formed of regenerated cellulose fiber and said stick is formed of plastic.
6. A test kit according to Claim 1 wherein said extraction reagent contains enzymes produced by the bacterium Streptomyces albus.
7. A test kit according to Claim 1 wherein said indicator reagent contains antibody bound to suspended latex particles which agglutinate when said antibody binds to said antigen, occurrence or non-occurrence of an antibody-antigen reaction being observed by agglutination or non-agglutination of said suspended particles.
8. A method for detecting Streptococcus A
comprising providing an applicator including an applicator stick and a fibrous swab and swabbing a biological sample with said swab, providing an extraction reagent containing enzymes for effecting release of Streptococcus A
antigen from said swab, dipping said swab in said extraction reagent and incubating said swab within said extraction reagent for a period of time suffi-client to release antigen from said fibrous swab, providing indicator reagent containing antibody reactive with said antigen and adding an Alcott of said extraction reagent thereto, and noting the occurrence or non-occurrence of an antibody antigen reaction, indicating the presence or absence of Streptococcus A in said biological sample.
comprising providing an applicator including an applicator stick and a fibrous swab and swabbing a biological sample with said swab, providing an extraction reagent containing enzymes for effecting release of Streptococcus A
antigen from said swab, dipping said swab in said extraction reagent and incubating said swab within said extraction reagent for a period of time suffi-client to release antigen from said fibrous swab, providing indicator reagent containing antibody reactive with said antigen and adding an Alcott of said extraction reagent thereto, and noting the occurrence or non-occurrence of an antibody antigen reaction, indicating the presence or absence of Streptococcus A in said biological sample.
9. A method according to Claim 8 wherein said swab is formed of regenerated cellulose fiber.
10. A method according to Claim 8 wherein said stick is formed of a synthetic material.
11. A method according to Claim 8 wherein said stick is formed of non-porous material.
12. A method according to Claim 8 wherein said swab is formed of regenerated cellulose fiber and said stick is formed of plastic.
13. A method according to Claim 8 wherein said extraction reagent contains enzymes produced by the bacterium Streptomyces album.
14. A method according to Claim 8 wherein said indicator reagent contains antibody bound to sus-penned latex particles which agglutinate when said antibody binds to said antigen, occurrence or non-occurrence of an antibody-antigen reaction being observed by agglutination or non-agglutination of said suspended particles.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US584,175 | 1984-02-27 | ||
| US06/584,175 US4618576A (en) | 1984-02-27 | 1984-02-27 | Diagnostic test for Streptococcus A |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1227131A true CA1227131A (en) | 1987-09-22 |
Family
ID=24336209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000466006A Expired CA1227131A (en) | 1984-02-27 | 1984-10-22 | Diagnostic test for streptococcus a |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US4618576A (en) |
| EP (1) | EP0153477B1 (en) |
| JP (1) | JPS60188847A (en) |
| AU (1) | AU580971B2 (en) |
| CA (1) | CA1227131A (en) |
| DE (1) | DE3465110D1 (en) |
| DK (1) | DK161219C (en) |
| FI (1) | FI79614C (en) |
| MY (1) | MY101456A (en) |
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| CN104644693B (en) * | 2015-02-16 | 2018-08-14 | 国药集团鲁亚（山东）制药有限公司 | The preparation method of injection A group streptococcus drugs |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3368549A (en) * | 1964-10-19 | 1968-02-13 | S E Massengill Company | Diagnostic swabs |
| US3790447A (en) * | 1972-07-05 | 1974-02-05 | Abbott Lab | Streptococci diagnostic method |
| US4203724A (en) * | 1976-08-16 | 1980-05-20 | Mitsubishi Chemical Industries Limited | Method and apparatus for the measurement of antigens and antibodies |
| JPS5742634A (en) * | 1980-08-27 | 1982-03-10 | Seikagaku Kogyo Co Ltd | Latex sensitized with group polysaccharide antibody in streptococcus hemolyticus |
| US4497900A (en) * | 1982-04-12 | 1985-02-05 | Abbott Laboratories | Immunoassay for Neisseria gonorrhoeae antigens |
| EP0109012A3 (en) * | 1982-11-12 | 1984-08-08 | Abbott Laboratories | Determination of streptococci |
| US4525452A (en) * | 1983-01-24 | 1985-06-25 | Btc Diagnostics Limited Partnership | Enzyme immunoassay with step of immersing sample in deionized water |
| AU2346684A (en) * | 1983-04-18 | 1984-11-07 | Quidel | Removal of self-binding and staph a cross-reactivity of anti-strep antibody |
| US4582810A (en) * | 1983-09-30 | 1986-04-15 | Becton, Dickinson And Company | Immuno-agglutination particle suspensions |
-
1984
- 1984-02-27 US US06/584,175 patent/US4618576A/en not_active Expired - Fee Related
- 1984-10-22 CA CA000466006A patent/CA1227131A/en not_active Expired
- 1984-10-26 AU AU34741/84A patent/AU580971B2/en not_active Ceased
- 1984-11-28 FI FI844676A patent/FI79614C/en not_active IP Right Cessation
- 1984-12-15 EP EP84115556A patent/EP0153477B1/en not_active Expired
- 1984-12-15 DE DE8484115556T patent/DE3465110D1/en not_active Expired
- 1984-12-28 JP JP59281851A patent/JPS60188847A/en active Granted
-
1985
- 1985-02-15 DK DK072085A patent/DK161219C/en not_active IP Right Cessation
-
1987
- 1987-09-29 MY MYPI87002124A patent/MY101456A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60188847A (en) | 1985-09-26 |
| FI79614B (en) | 1989-09-29 |
| EP0153477A1 (en) | 1985-09-04 |
| DK161219B (en) | 1991-06-10 |
| MY101456A (en) | 1991-11-18 |
| FI844676L (en) | 1985-08-28 |
| EP0153477B1 (en) | 1987-07-29 |
| JPH0315147B2 (en) | 1991-02-28 |
| FI79614C (en) | 1990-01-10 |
| DK72085D0 (en) | 1985-02-15 |
| FI844676A0 (en) | 1984-11-28 |
| DK161219C (en) | 1991-11-25 |
| US4618576A (en) | 1986-10-21 |
| AU580971B2 (en) | 1989-02-09 |
| AU3474184A (en) | 1985-09-05 |
| DE3465110D1 (en) | 1987-09-03 |
| DK72085A (en) | 1985-08-28 |
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